The overall goal of this proposal is to characterize the phospholipid binding specificity of factor IX. Factor IXa cleaves factor X to produce Xa in a vitamin K-dependent reaction. The absence of factor Ix causes hemophilia B. The factor IXa reaction is one of many in the coagulation cascade requiring a membrane surface for assembly of the enzyme, substrate, and cofactor. The central hypothesis of this proposal is that phospholipid composition affects the binding of factor IX and the factor IXa catalyzed conversion of factor X to factor Xa. The effect of phospholipid composition on the enzymatic function of factor IXa will be examined by measuring factor Xa generation in the presence of phospholipid vesicles and soluble phospholipids of varying compositions. In addition, the binding specificity of factor DC for phospholipid vesicles will be determined using light scattering. Identification of the portions of the phospholipids important in the binding will be discovered by using soluble phospholipids to compete with phospholipid vesicles for the binding of fluorescently labeled factor IX in flow cytometry experiments. These studies will be complemented by 1H NMR spectroscopy experiments examining molecular contacts between factor IX( 147) and l ,2-dihexanoic-sn-glycero~3 phospho-L-serine. Ultimately, the structure of the factor IX(l47)-phosphatidylserine complex will be determined by two- dimensional NMR analysis. The results of this proposal will lead to new information about how phospholipid membranes contribute to the vitamin K-dependent coagulation reactions.